For many tissues of the body, ordered growth requires that the balance between cell production, cell differentiation and cell death is precisely regulated. Among the best studied of the rapidly-turning over tissues is the haematopoietic system, in which a multitude of terminally-differentiated cell types is generated from a relatively small number of stem cells and committed progenitor cells. Many of the factors which participate in the regulation of this process are known, thanks to data largely generated using in vitro systems. Recently, it has been possible to use gene targeting in embryonic stem cells (ES cells) to generate mice deficient in growth regulators, in order to examine whether these growth regulators indeed play a pivotal role in the regulation of normal blood cell production. See International Patent Application No. WO 95/23862 (PCT/AU94/00103).
Cells of the immune system are subject not only to regulation by a variety of growth factors and cytokines, but also utilise a complex system of signal transduction to mediate cell activation following antigen stimulation.
In particular, the src family of protein tyrosine kinases has been implicated in cell signalling through the physical association of these kinases with different cell surface receptors which on their own lack intrinsic catalytic activity (Bolen et al, 1992). Like several other members of the src family, the protein tyrosine kinase known as lyn is expressed in a broad range of cell types and tissues (Bolen et al, 1992). Largely through co-precipitation studies, lyn has been shown to be physically associated with a number of haematopoietic cell surface receptors, including the B cell antigen receptor (BCR) (Yamanashi et al, 1991; Burkhardt et al, 1991; Campbell and Sefton, 1992), CD40 (Ren et al, 1994), the lipopolysaccharide (LPS) receptor (Stefanova et al., 1993), the high affinity Fc.epsilon.RI complex (Eiseman and Bolen, 1992), and the G-CSF receptor (Corey et al, 1994). In most cases, more than one member of the src family has been found to be associated with the same cell surface receptor, raising the possibility of functional redundancy within the src family. This notion is supported by the milder than expected phenotype shown by mice in which one or other src-related kinase genes has been disrupted by homologous recombination in embryonic stem (ES) cells (reviewed in Varmus and Lowell, 1994). However, mice in which the lyn gene is disrupted have not hitherto been described.
A competent, signal-transducing BCR consists of an antigen-binding membrane immunoglobulin (Ig) non-covalently associated with disulphide-linked heterodimers of Ig-.alpha. and Ig-.beta./.gamma. subunits (Reth, 1992). While the molecules that make up this BCR complex lack intrinsic catalytic activity, stimulation of resting B cells with antibodies to membrane Ig induces rapid tyrosine phosphorylation of B cell proteins, suggesting associated tyrosine kinase activities (Gold et al, 1990; Campbell and Sefton, 1990; Gold et al, 1991). This increase in total cellular tyrosine phosphorylation is correlated with an increase in the enzymatic activity of several members of the src family, including lyn, blk, fyn, and fgr; indeed, co-immunoprecipitation studies have shown a physical association between the BCR complex and several members of the src family (Yamanashi et al, 1991; Burkhardt et al, 1991; Campbell and Sefton, 1992; Wechsler and Monroe, 1995). A highly conserved motif, termed the immunoreceptor tyrosine-based activation motif (ITAM), is found in many signal transducing subunits, including the cytoplasmic domain of the Ig-.alpha. and Ig-.beta./.gamma. molecules. Conserved tyrosine residues within this ITAM are a target for phosphorylation upon ligation of the BCR, and presumably provide docking sites for additional molecules involved in B cell signalling, such as PI 3-kinase, PLC-.gamma.2 and GTPase-activating protein. Recent studies have shown that the cytoplasmic domain of the Ig-.alpha. chain is constitutively associated with the src family kinases lyn and fyn (Clark et al, 1992; Pleiman et al., 1994a). This suggests that members of the src family may directly phosphorylate the ITAMs, and thus participate in very early events in the BCR signal transduction cascade.
Signalling events from the BCR resemble those thus far characterised for the Fc.epsilon.RI complex (Ravtetch, 1994). Fc.epsilon.RI is a tetrameric structure consisting of a ligand binding .alpha. subunit, a .beta. subunit and homodimeric .gamma. subunits (Blank et al, 1989). Like the Ig.alpha. and Ig.beta./.gamma. signalling molecules of the BCR complex, the cytoplasmic domains of the .beta. and .gamma. subunits of Fc.epsilon.RI also contain ITAMs (Ravetch, 1994). Biochemical studies have shown that lyn is associated with the .beta. subunit, and it is thought that on Fc.epsilon.RI triggering, lyn becomes activated and phosphorylates critical tyrosine residues in the ITAMs of both the .beta. and .gamma. subunits. The phosphorylation of the .gamma. subunit recruits and activates p72syk, which in turn activates other molecules involved in the signal transduction cascade (reviewed in Ravetch, 1994).
To gain an insight into the physiological role of lyn and to gauge its importance in relaying signals from these different cell surface receptors, we have generated mice which are unable to express lyn (lyn -/- mice) by gene targeting in ES cells. Our results show that lyn is an indispensable component of the BCR and Fc.epsilon.RI complexes, and that its actions are required for the elimination of autoreactive antibodies. In addition, our longitudinal studies of lyn -/- mice show that the absence of the lyn gene is associated in the long term with depletion of lymphoid tissue, extramedullary haematopoiesis, expansion of cells of the myeloid lineage, glomerulonephritis leading to renal failure, and lesions in spleen, lymph node, liver and kidney resembling malignancy. Consequently the lyn -/- mouse is useful as a model of autoimmune disease, especially autoimmune glomerulonephritis, and of certain malignancies or dysplasias of myeloid origin, such as myeloid leukemia, malignant histiocytoma, and histiocytosis.